Project Summary Electron-transferring flavoproteins (ETFs) are a large and broadly distributed family of electron carriers that play critical roles in a variety of metabolic processes, including b-oxidation of fatty acids in humans. Defects in ETFs give rise to several specific disease states. A subset of the ETF family constitute a recently discovered class of complex flavoproteins that catalyze the coupled reduction of high- and low-potential electron acceptors using a median-potential electron donor in a process referred to as electron bifurcation. An example of such a bifurcating ETF is the EtfABCX system from Pyrobaculum aerophilum. This enzyme accepts reducing equivalents from NADH, passing the reducing equivalents on to the high-potential menaquinone and the low-potential iron- sulfur protein ferredoxin in a manner that is overall thermodynamically favorable. The proposed work will examine in detail the kinetics of electron bifurcation as catalyzed by EtfABCX, critically evaluating the involvement of protonation/deprotonation processes associated with the primary electron transfer events.